Background: Fragile X mental retardation syndrome (FXS), is the most common cause of intellectual disability and the most common known cause of autism. Other symptoms of FXS include depression, sensory processing deficits, aggressive behavior, connective tissue problems and digestive difficulties. This disorder arises when the number of CGG-repeats in the 5 UTR of the FMR1 gene exceeds 200. Such alleles become silenced. This results in a deficiency of the protein product of this gene, FMRP, which is involved in the regulation of translation of a subset of mRNAs. The FMRP deficiency in brain results in aberrant dendritic spine morphology and defects in the response to synaptic activation. The mechanism of gene silencing is unknown.[unreadable] [unreadable] Progress report: We have taken 2 broad approaches. One has been to understand the factors important for normal FMR1 expression (Kumari and Usdin, 2001, 2005) and for the expression of an autosomal homolog of FMR1, FXR2, which encodes a protein whose function is thought to partially overlap with FMRP (Mahishi and Usdin, 2006). [unreadable] [unreadable] The 2nd approach as been to study the molecular events leading to gene silencing on FXS alleles. Previous work has demonstrated that these alleles are heavily methylated. A DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (5-aza-dC), has been shown to partially reverse the FMR1 silencing. However, not only is this compound toxic, but it requires DNA replication to be effective. It is thus not likely to be useful in post-mitotic cells like neurons where the FMRP deficiency is most apparent. We have shown that deacetylation of Histone H4K16 is a late downstream consequence of DNA methylation (Biacsi, Kumari and Usdin, 2008). We have also shown that deacetylation is carried out by SIRT1, a protein deacetylase. Inhibition of this enzyme is possible using nicotinamide or splitomicin and treatment of patient cells allows reactivation of the FMR1 gene. This reactivation does not require DNA demethylation and thus SIRT1 inhibitors may be useful for FMR1 gene reactivation in neurons. [unreadable] [unreadable] We have shown that in addition to the DNA methylation pathway, silenced alleles are also targets of the Polycomb-mediated pathway of gene silencing. Paradoxically, levels of the repressive chromatin mark, H3K27Me3, that is deposited by these proteins, rises as the FMR1 gene is reactivated. This rise occurs in parallel with a rise in the levels of a mark of active chromatin, H3K4Me2. Thus reactivation of the silenced allele generates a bivalent chromatin state. Such states are thought to predispose affected genes to subsequent gene silencing. Work is underway to try and understand the mechanism responsible.